Difference between revisions of "Team:TNCR Korea/Results"

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{{TNCR_Korea}}
 
 
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<title>TNCR_KOREA &mdash; Digestable Gluten</title>
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<h1>Results</h1>
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<p>Here you can describe the results of your project and your future plans. </p>
 
  
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<h5>What should this page contain?</h5>
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<ul>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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<h5>You should also describe what your results mean: </h5>
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<link rel="stylesheet" type="text/css" href="https://2017.igem.org/Template:TNCR_Korea_style/CSS?action=raw&ctype=text/css">
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
 
  
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<h5>Inspiration</h5>
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<p>See how other teams presented their results.</p>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<p>We had to confirm that recombinant plasmid produced DPP4 protein and had weak, moderate, high expression for respective vector. In order to check if our recombinant plasmid works under realistic condition, inside mammalian cell, we performed transfection and western blotting. </p>
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<p>Recombinant plasmid was inserted into HEK293T cell. With DNA transfection reagent, jetPEI(from Polyplus), we performed transfection of recombinant plasmid to mammalian cell. After certain that plasmid was successfully transfected into the cell, we moved on to verify the expression of DPP4 of each cell. </p>
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<p>We used two IB, Flag-DPP4(1:1000 ratio) and beta-actin(1:5000 ratio). mAB ANTI beta-actin was used as internal control for western blotting. This allows equal expression of the gene in cells ubiquitously. In the picture, the expression is on 55kDa with similar band size for weak, moderate, and high(They are relatively similar). Then to observe the difference in expression regarding the Anderson promoter that controls the gene expression, we used mAB ANTI-FLAG M2. We could have used DPP4 antibody but as the vector already contains FLAG, we decided to utilize FLAG antibody to check the vector’s expression. The band on the top is shown at 100kDa and have significantly distinguishable band sizes. When we compare the size of the bands of each weak, moderate, and high, we can observe that weak has the least expression, moderate in between weak and strong, and high has the most expression. This demonstrates that our recombinant plasmid successfully functions inside the mammalian cell, not only producing DPP4 but also regulating the amount of its expression. </p></br></br></br>
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<p>Finally, we wanted to test the possibility of application for further study. After acquiring several individual feces, we isolated the gut bacteria and compared their composition with RFLP methods.  Here is one of the significantly different phenomenon.</p>
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<center><img src="https://i.imgur.com/XLbBpsB.png" alt="TNCR_Korea Team" class="img-responsive" height="500" width="700"></center>
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Latest revision as of 03:04, 2 November 2017

TNCR_KOREA — Digestable Gluten

Project

Results

We had to confirm that recombinant plasmid produced DPP4 protein and had weak, moderate, high expression for respective vector. In order to check if our recombinant plasmid works under realistic condition, inside mammalian cell, we performed transfection and western blotting.

Recombinant plasmid was inserted into HEK293T cell. With DNA transfection reagent, jetPEI(from Polyplus), we performed transfection of recombinant plasmid to mammalian cell. After certain that plasmid was successfully transfected into the cell, we moved on to verify the expression of DPP4 of each cell.

We used two IB, Flag-DPP4(1:1000 ratio) and beta-actin(1:5000 ratio). mAB ANTI beta-actin was used as internal control for western blotting. This allows equal expression of the gene in cells ubiquitously. In the picture, the expression is on 55kDa with similar band size for weak, moderate, and high(They are relatively similar). Then to observe the difference in expression regarding the Anderson promoter that controls the gene expression, we used mAB ANTI-FLAG M2. We could have used DPP4 antibody but as the vector already contains FLAG, we decided to utilize FLAG antibody to check the vector’s expression. The band on the top is shown at 100kDa and have significantly distinguishable band sizes. When we compare the size of the bands of each weak, moderate, and high, we can observe that weak has the least expression, moderate in between weak and strong, and high has the most expression. This demonstrates that our recombinant plasmid successfully functions inside the mammalian cell, not only producing DPP4 but also regulating the amount of its expression.








Finally, we wanted to test the possibility of application for further study. After acquiring several individual feces, we isolated the gut bacteria and compared their composition with RFLP methods. Here is one of the significantly different phenomenon.

TNCR_Korea Team